The development of a vaccine for syphilis has been hindered by inability to culture the causative organism Treponema pallidum in vitro. Reports of successes have been published, but when identical procedures were attempted by different investigators, multiplication was not detected. In order to achieve the necessary cultivation, it is essential that a method be reproducible in other laboratories before it can be called successful. Thus, see the following U.S. Pat. Nos. 2,255,079, 2,513,327, 2,709,670, 3,502,546 and 4,098,646. Additionally, see Graves et al, Retention of Motility and Virulence of T. pallidum in vitro; Infection and Immunity. 1975, 12(5) 1116-20 (U.S.A.),--Serzhantova, Growth of T. pallidum, in a Thioglycollate Medium Vestn. Dermatol. 1969, 43(8) 48-51 (Russian),--Boak et al, Studies on the Cultivation of T. pallidum, American Journal of Syphillis, Gonorrhea, Venereal Diseases 33, 409-15 (1942). See also Fitzgerald, The Future of Tissue Culture Methods for Growth of Treponema pallidum in vitro, Sexually Transmitted Diseases April-June, 1980, pp 97-99,--and Musher et al.--The Role of A Vaccine for Syphilis--ibid October-December 1977, pp 163-166, Cox, C. D. and M. K. Barber. 1974. Oxygen uptake by Treponema pallidum. Infect. Immun. 10:123-127.
Fieldsteel, A. H., F. A. Becker, and J. G. Stout. 1977. Prolonged survival of virulent Treponema pallidum (Nichols strain) in cell-free and tissue culture systems. Infect. Immun. 18:173-182.
Fieldsteel, A. H., D. L. Cox, and R. A. Moeckli. 1981. Cultivation of virulent Treponema pallidum in tissue culture. Infect. Immun. 32:905-915.
Fieldsteel, A. H., J. G. Stout, and F. A. Becker. 1981. Role of serum in survival of Treponema pallidum in tissue culture. In Vitro 17:28-32.
Fitzgerald, T. J., R. C. Johnson, J. A. Sykes, and J. N. Miller. 1977. Interaction of Treponema pallidum (Nichols strain) with cultured mammalian cells: Effects of oxygen, reducing agents, serum supplements, and different cell types. Infect. Immun. 15:444-452.
Kimm, G. E., R. H. Allen, M. J. Morton, and J. F. Morgan. 1962. Studies on the in vitro survival of virulent Treponema pallidum. Am. J. Hyg. 75:339-356.
Nelson, R. A., Jr. 1948. Factors affecting the survival of Treponema pallidum in vitro. Am. J. Hyg. 48:120-132.
Sandok, P. L., H. M. Jenkin, H. M. Mattews, and M. S. Roberts. 1978. Unsustained multiplication of Treponema pallidum (Nichols virulent strain) in vitro in the presence of oxygen. Infect. Immun. 19:421-429.
Turner, T. B. and D. H. Hollander. 1957. Biology of the treponematoses. W.H.O. Monogr. Ser. No. 35.
Weber, M. M. 1960. Factors influencing the in vitro survival of Treponema pallidum. Am. J. Hyg. 71:401-417.
As an almost natural sequel to the discovery that Treponema pallidum is the causative agent of syphilis, attempts were made to cultivate it. Although many investigators have claimed to have cultivated it in vitro, none of the isolated strains have been pathogenic or have been shown to have a causal relationship to treponemal disease in humans. Furthermore, investigators who carried out serologic studies on the cultivable Nichols, Noguchi, Reiter, Kazan, and Kroo strains of supposed T. pallidum found that these strains fell into three distinct serological groups and that they were morphologically and antigenically different from T. pallidum. Hence they questioned the identification of these treponemes as strains of T. pallidum. More recently, the inventors herein utilized biochemical analysis to determine whether there are genetic relationships between virulent T. pallidum and five avirulent cultivable treponemes isolated from humans. Not only did the deoxyribonucleic acid (DNA) base composition (G+C content) of the cultivable treponemes differ markedly from that of T. pallidum, but saturation reassociation assays revealed no detectable DNA sequence homology. Consequently, the nonpathogenic cultivable treponemes studied were neither variants nor mutants of T. pallidum and were actually genetically distinct organisms.
Originally it had been generally assumed that T. pallidum was an anaerobic organism because it survived poorly under aerobic conditions. Therefore, in attempts to obtain survival and/or replication of T. pallidum in vitro, anaerobic conditions were maintained and reducing agents were added to media to lower the oxidation-reduction potential. Although the 50% survival time (ST.sub.50) of T. pallidum under these conditions was as long as 16 days, there apparently was no correlation between the ST.sub.50 and virulence for rabbits, which was lost after 6 days in vitro.
Despite the apparent anaerobic nature of treponemes, the presence of cytochromes has been demonstrated in the nonpathogenic cultivable Reiter treponeme (T. phagedenis biotype Reiter) which suggested possible utilization of molecular oxygen under certain conditions. More recently, it has been shown that T. pallidum consumed oxygen at the same rate as did a known aerobic spirochete, Leptospira. The oxygen uptake of the former was cyanide-sensitive, indicating that T. pallidum contained a functioning cytochrome oxidase system and therefore should be capable of aerobic respiration. In a study on the mechanisms involved in terminal electron transport by T. pallidum, these observations were confirmed, leaving little doubt as to the capabilities of virulent T. pallidum for aerobic respiration. Therefore, in view of the failure of all attempts to cultivate T. pallidum under anaerobic conditions, it appeared more than likely that oxygen is a requirement for in vitro replication.
Between 1913 and 1948, sporadic attempts were made to cultivate T. pallidum in tissue culture of mammalian tissues, with uniformly negative results. Recently, there has been renewed interest in the cultivation of T. pallidum in mammalian cell cultures. Several investigators, using both aerobic and anaerobic culture systems, have reported the attachment of T. pallidum to tissue culture cells, with periods of survival ranging from 24 hours to 23 days. Survival appeared to be best under conditions of low oxygen tension (3%) and when reducing agents were added to the medium. However, there was little or no evidence that the treponemes had replicated.
For example, in 1976, Jones et al. (British Journal of Venereal Diseases 52:18-23) claimed to have demonstrated replication and subculture of pathogenic T. pallidum in cultures of baby hamster kidney, but Foster et al. (ibid) 53:338-339 failed to corroborate these results. Others demonstrated what they called "unsustained multiplication" of T. pallidum in cultures under reduced oxygen tension, both in the presence and absence of mammalian cells. Virulence of the treponemes decreased as a function of time in vitro.